Method for Treating Arteriosclerosis

ABSTRACT

The invention relates to a method for identifying substances with antiapoptotic activity, where
         i) cells which express both CD47 and the integrin α v β 3  are cultured,   ii) the cells are caused to produce an apoptosis-inducing substance, and/or a substance or substances which induces/induce apoptosis is/are added,   iii) the test substance is added, and   iv) the apoptosis rate is measured,
           and to the substances which can be identified with the method.

This is a division of co-pending U.S. patent application Ser. No.11/327,095 filed Jan. 6, 2006, which is a division of U.S. patentapplication Ser. No. 10/129,295, now U.S. Pat. No. 7,008,773 B1 issuedMar. 7, 2006, which is the U.S. national phase of internationalapplication No. PCT/EP00/10806 filed Nov. 2, 2000, which claims priorityto DE 199 52 960.4 filed Nov. 3, 1999, the entire respective disclosuresof which are hereby incorporated by reference.

The invention relates to a method for identifying substances withantiapoptotic activity and to the substances identified thereby. Theinvention further relates to pharmaceutical preparations which comprisesuch substances, and to their use for the treatment of vasculardisorders.

Arteriosclerosis (“arterial calcification”) is the most important andcommonest pathological change in the arteries. It is associated with achange in the vessel content and with lesions of the endothelium (of theendothelial cells) and, caused thereby, metabolic and cellular reactionsof the vessel wall. Disturbances of arterial blood flow are thecommonest cause of death in industrialized countries (about 50%). Thebasis thereof in most cases is arteriosclerosis.

The efficacy of agents currently employed against coronary heart diseaseis based essentially on reducing the myocardial oxygen consumption andadjusting the reduced coronary blood flow. These agents additionallybring about dilation of the coronary arteries. On the other hand, it isscarcely possible by medication to increase the coronary blood flow incases of arteriosclerotic coronary stenosis because the diseased vesselscan no longer be dilated. All medicaments act only in a very late stageof the disorder. No agents are yet known to be able to treat the causeof the disorder directly or to be suitable for early diagnosis. In theabsence of an effective possibility of early diagnosis, most patientsare treated only after a mild cardial infarction. In the advanced stage,usually a surgical operation (e.g. bypass) is the only option left.

The inner walls of all blood vessels are lined by endothelial cells.They are involved in the regulation of various physiological processessuch as, for example, the regulation of blood pressure and thedegeneration and regeneration of vessels. A large number of pathologicalsituations is associated with dysfunction of endothelial cells, forexample the focal development of arteriosclerosis.

Apoptosis (synonym: programmed cell death) is an irreversible processand cannot be stopped. Accordingly, an apoptotic cell inevitably dies.

European patent application EP-A-0 903 149 describes a method foridentifying apoptosis-inducing substances in immune cells. It showedthat substances which bind to the integrin-associated protein (IAP or CD47) on the surface of immune cells may have the ability to induceapoptosis. The mechanism of action was not described.

It has already been proposed that IAP is involved in the formation of aspecific calcium channel (Schwartz, M. A. et al., The Journal ofBiological Chemistry, 268:27, 19931-19934). A role of this hypotheticalcalcium channel in the induction of apoptosis was not mentioned.

Arteriosclerotic lesions form more frequently at bifurcations (forks) inthe vascular system than in unbranched regions of the blood vessels. Ithas already been possible to observe apoptotic endothelial cells in theregion of these lesions. It is suggested that apoptotic endothelialcells are involved in the development of arteriosclerosis [Asakura, T.,Karino, T., Circulation Research, 66, 1045-1066 (1990)].

No agents are currently known for the prevention or therapeutictreatment of the occurrence of apoptosis in endothelial cells of thevascular system.

The present invention is therefore based on the object of providing amethod with which it is possible to find substances which inhibitapoptosis in endothelial cells. This method is simple to operate andreliable and cost-effective to carry out. The substances identified inthis way are to be employed as ingredients of pharmaceuticalpreparations for the treatment of conditions in which an inhibition ofapoptosis is indicated, in particular of vascular disorders,particularly preferably of arteriosclerosis.

This object is achieved by a method for identifying apoptosis-inhibitingsubstances and substances with antiapoptotic activity, wherein cellswhich express both IAP and the integrin α_(v)β₃ are cultivated, and/orthe cells are caused to produce an apoptosis-inducing substance and/or asubstance(s) which induces/induce apoptosis is/are added, a testsubstance is added and the apoptosis rate is measured. The inventionlikewise encompasses substances which can be identified by the claimedmethod, pharmaceutical preparations which comprise such a substance asactive ingredient, and the use of this pharmaceutical preparation forthe treatment of vascular disorders, in particular for the treatment ofarteriosclerosis. The invention further encompasses the use ofsubstances which have been identified with the aid of the method of theinvention for treating vascular disorders, in particular for treatingarteriosclerosis.

The inventors have surprisingly shown that the simultaneous binding ofthrombospondin-1 (TSP-1) to IAP and integrin α_(v)β₃ induces apoptosisin endothelial cells. It has additionally been possible to show,surprisingly, that TSP-1 is produced by the endothelial cellsthemselves, and thus the apoptosis is self-induced or spontaneous. Theseinvestigations were carried out in conventional, static cell cultures.These are distinguished by the absence of any flows in the cell culturemedium. However, it was unexpectedly possible to show that endothelialcells produce no TSP-1 in a perfusion culture, i.e. under conditionswhere the cells are confronted by a flowing cell culture medium, andapoptosis occurs to only a very small extent or not at all in this cellculture.

Supplementation of fresh medium with TSP-1 causes an increase inspontaneous apoptosis in statically cultivated endothelial cells. Thisincrease corresponds approximately to the effect of staticallyconditioned medium (i.e. medium which has previously been used for thecultivation of HUVEC in static culture) (table 1). This shows thatstatically conditioned medium has the ability to induce apoptosis via amediator such as TSP-1.

The term “conditioned” medium means herein a cell culture medium whichhas previously been used for cultivating other cells. This medium isdistinguished by having dissolved in it soluble mediators, e.g. growthfactors, hormones etc., which are produced by cells during theircultivation.

It was possible to show by use of an anti-TSP-1 antibody which binds toand thus neutralizes TSP-1 that TSP-1 is the mediator of the apoptosisof endothelial cells. The effect of added TSP-1 can be suppressed, justlike the effect of statically conditioned medium, by addition of apolyclonal antiserum against TSP-1 and by addition of a monoclonalanti-TSP-1 antibody (table 1).

TABLE 1 Apoptosis Culture rate conditions Culture medium (24 h) (%)Static Fresh medium (a) 0.9 ± 0.1 Static Conditioned medium (b) 3.0 ±0.2 Static Conditioned + anti-TSP 1 (c) 0.1 ± 0.1 Static Fresh + TSP 1(e) 3.0 ± 0.4 Static Fresh + anti-TSP 1 (d) 0.2 ± 0.1 Static Fresh + TSPI + anti-TSP 1 (f) 0.2 ± 0.1 Static Fresh + mAb TSP 1 (g) 0.4 ± 0.1

The apoptosis rate (%) was determined as shown in example 6. The letters(a) to (g) relate to the investigations presented in example 10 and tothe specific experimental conditions chosen therein (see example 10).

It was additionally shown qualitatively by Western blot investigationsthat TSP-1 is secreted only by statically cultivated endothelial cells(FIG. 1). The secretion rates were determined for dynamic and staticpostconfluent cultures (FIG. 2). The results showed that induction ofapoptosis can be controlled via the TSP-1 secretion rate.

The term “static cell culture (conditions)” means here a cultivation ofcells under conditions with which invariable, i.e. consistentlydirected, laminar flows do not occur in the cell culture mediumsurrounding the cells. The “static cell culture conditions” in thissense used herein thus include cell culture conditions under whichturbulent or variable laminar flows, that is to say, for example, thosewith changing directions of flow or even with reversal of flow, occur.The term “dynamic cell culture (conditions)” means here cell cultureconditions with which only consistently directed, laminar flowconditions prevail in the cell culture medium, i.e. cell cultureconditions like those which can be achieved in the prior art for examplewith the aid of a so-called perfusion culture. It is clear that theconditions of idealized physical fluid mechanics are not attained eitherin the blood vessel system, i.e. in the in vivo situation, or under cellculture conditions. The flow conditions described herein are thus thosewhich can approximately be attained, and are used, generally in cellculture experiments in the prior art.

The shear stresses thus acting on the cultivated cells vary with theflow conditions. A consistently directed laminar flow results in a shearstress which is greater than 0.001 dyn/cm² and whose vector sum isgreater than under variable flow conditions with changing directions offlow. Static cell culture (conditions) are distinguished by a distinctlysmaller (<0.001 dyn/cm²) or absolutely no stress. Dynamic cell culturesshow shear stresses of >0.001 dyn/cm² or a Reynolds number of >0.1.Turbulent flows may occur at Reynolds numbers of >200 (−1000) (dependingon the geometry of the flow chamber) and, like static or variable flowconditions, no longer have a protective character in relation toinduction of apoptosis.

However, surprisingly, addition of TSP-1 to a perfusion culturegenerally has no effect on apoptosis. This shows that apoptosis dependsnot only on the occurrence of TSP-1 in the bloodstream but, on thecontrary, also on the occurrence or accessibility of specific receptorson the surface of the cells. It was surprisingly possible to show inthis connection that the integrin α_(v)β₃ receptor is detectable onstatically and dynamically cultivated cells, whereas the IAP receptor isexpressed in detectable quantities only in static culture.

It is known that TSP-1 binds to the integrin α_(v)β₃. The binding ofTSP-1 to the α_(v)β₃ integrin is mediated by an RGD sequence motif. Apotent agonist of RGD-mediated binding, acyclic Arg-Gly-Asp-(D-Phe)-Valpeptide (cyclic RGD peptide) (supplied by Merck), was therefore added topostconfluent endothelial cells. However, this unexpectedly had noeffect on the apoptosis rate. This showed that binding of TSP-1 to theintegrin α_(v)β₃ at least does not on its own lead to apoptosisinduction (table 2).

TABLE 2 Culture medium (48 h) Apoptosis rate (%) Fresh medium (control)1.5 ± 0.2 Conditioned medium 6.4 ± 0.5 Fresh + TSP1 6.9 ± 1.2 Fresh +active RGD 1.2 ± 0.5 Fresh + inactive RGD 1.0 ± 0.4 Fresh + CBD 2.0 ±0.2 Fresh + CBD + active RGD 7.0 ± 0.6 Fresh + CBD + inactive RGD 1.7 ±0.6 Fresh + CBD + active RGD + anti-TSP1 6.3 ± 1.3

The apoptosis rate was determined as shown in example 5. Cultivation infresh medium serves as control and shows a spontaneous apoptosis rate instatic cell culture. Conditioned medium has previously been incubatedwith endothelial cells for 48 to 72 hours, so that it contained thefactors secreted by the endothelial cells. The apoptosis rate caused bythe conditioned medium was at the same level as with fresh medium+TSP-1,which showed that one or more mediator(s) of apoptosis must have beenpresent in this conditioned medium. The preparation of conditionedmedium is explained in example 5.

Another possible interaction of TSP-1 with a receptor on endothelialcells is the binding to IAP via the C-terminal cell binding domain(CBD). The C-terminal cell binding domain (CBD) is a TSP-1 domain whichinteracts specifically with IAP. A truncated TSP-1 which consists onlyof this C-terminal cell binding domain is marketed by Bachem as CBDpeptide. Addition of the CBD peptide did not lead to an increase in theapoptosis rate (table 2). Simultaneous addition of the CBD peptide andthe cyclic RGD peptide did, however, surprisingly lead to a markedincrease in the apoptosis rate (table 2), which was at a similar levelto the increase in the apoptosis rate by addition of TSP-1. It followsfrom this that only simultaneous binding to IAP and the integrin α_(v)β₃is effective for apoptosis.

Substitution of an inactive RGD peptide for one of the other twopeptides causes the effect to disappear again. Giving anti-TSP-1antiserum simultaneously with the two active peptides has no effect onthe increase in the apoptosis rate (table 2). This shows that theproduction of TSP-1 during the incubation has no effect. It was thuspossible to show, surprisingly, that the activity of TSP-1 for inducingspontaneous apoptosis is mediated by simultaneous binding to IAP andα_(v)β₃.

Binding of TSP-1 to IAP and α_(v)β₃ is followed by a calcium influx intothe cell (FIG. 3). This calcium influx is observed immediately afteraddition (within a period of a few seconds) and is one step in thesignal transduction which finally leads to induction of apoptosis. It isthus possible by using fluorescent calcium indicators [Merrit, J. E. etal., Biochem. J. 269, 513-519 (1990)] to set up a very quick test methodfor inhibition of apoptosis through the nonappearance of the calciumsignal (FIG. 3, explained in detail in example 6).

In further investigations carried out by the inventors it was possibleto show not only that static culture conditions lead to induction ofapoptosis in appropriate cells, but that variable or turbulent flowconditions are sufficient therefor. Introduction of an impediment into aperfusion chamber leads to variable flow profiles. One model thereof isshown in FIG. 4. A typical flow chamber is distinguished by thepossibility of generating a liquid flow which is passed at defined flowrates through a flow chamber. The flow can be driven, for example, by apump or gravity. The particular liquid is passed via suitable flexibletubing or rigid tubes through the actual chamber. In the present system,the flexible tubing is of silicone and the rigid tubes are of stainlesssteel. The chamber is defined by a Petri dish with a polycarbonateinsert and silicone seals.

There are also other possibilities for generating a laminar flow. It isalso possible to employ for this purpose other suitable apparatus, e.g.a cone and plate apparatus, in which the flow is generated by therotation of a liquid-filled plate or the rotation of a cylindrical plugin the liquid on the plate.

It was further possible to show for the first time by in vitroinvestigations that apoptosis is induced in endothelial cells exactly inthe regions of variable flow profiles. It was possible to demonstratethis by a space-resolved investigation of apoptotic cells in theperfusion system (FIG. 5).

The presented results show that the actual cause of the induction ofapoptosis is the absence of an invariable laminar flow. This causes theendothelial cells to secrete TSP-1, which subsequently leads, throughthe interaction with the integrin α_(v)β₃ and IAP, to induction ofapoptosis.

It is possible on the basis of the presented findings to develop a testmethod which makes a targeted search for inhibitors of the induction ofapoptotic processes possible. It is thus possible to identify substanceswith antiapoptotic activity. For this purpose, cells which express bothIAP and the integrin α_(v)β₃ are cultured. The cells may be natural orgenerated by recombination. Such cells are known to the skilled worker.Preference is given in this connection to cultivated endothelial cellsand, in a particularly preferred embodiment, to human umbilical veinendothelial cells (HUVEC). Further examples of suitable cells aremonocytes, erythrocytes, activated T cells, fibroblasts, bloodplatelets, CHO cells, smooth muscle cells and ovarian tumor cells. Wholecell lines may also be suitable, such as, for example, bone marrow celllines. The cells used in the method of the invention may also be cellswhich have been genetically modified so that they express recombinantIAP or integrin α_(v)β₃, which has been modified where appropriate withretention of the original binding properties, on their cell surface.

These cells are cultivated in suitable cell culture vessels in suitablecell culture media, which are normally standard media generally known inthe prior art. For example, the cells are cultivated in DMEM, M-199, IFbasal media etc. Suitable culture media are now available for virtuallyall cells and cell lines. Growth factors and hormones such as, forexample, fetal calf serum (FCS) can be added to the culture mediumbefore starting the culture. Mammalian cells are usually cultivated at37° C. in a 5% CO₂ atmosphere which, in connection with the buffers usedin the cell culture media, e.g. sodium carbonate buffers, makes itpossible to stabilize the pH of the cell culture medium. A furtherpossibility is to add to the cell culture media, before starting thecultivation, antibiotics which interact specifically with prokaryoticcontaminating microorganisms and inhibit their growth, but leave thegrowth of the eukaryotic cells virtually unaffected and thus protect thecell culture from contamination. Further hints and information for cellculturing can be found in standard works, e.g. Zell- und Gewebekultur,3rd edition, Toni Lindl, Jörg Bauer, (1994), Gustav Fischcr Verlag.

Suitable culture conditions for cultivating HUVEC and other endothelialcells are given in example 1 and example 2.

Apoptosis is then induced by the culturing conditions or by adding oneor more apoptosis-inducing substances. Culturing conditions of this typeare preferably those with which regular laminar flows do not occur, asexplained above.

Mediators can be added to the test system in addition to or in place ofthe chosen flow conditions for induction or increasing the apoptosisrate. Simultaneous addition of mediators increases the sensitivity ofthe test system. The spontaneous apoptosis rate in a static cell cultureis relatively low compared with apoptosis rates induced by mediatorssuch as TNF-α. The apoptosis rates reached in the tests of thespontaneous apoptosis rate induced by the static cell culture conditionare in the range from 0.5 to 7% of the total cells. In the case ofapoptosis induced by mediators, apoptosis rates of up to 50% may bereached.

For this reason, in the method of the invention there is addition,particularly preferably before measurement of the inhibition of theapoptosis rate by potential inhibitors, of substances which induce anincreased apoptosis rate, so that the measured signal is enhanced. Theadded mediator preferably brings about apoptosis rates of from at least1% up to more than 10 to 50%. This particularly preferably entailsadding TSP-1 to the cell culture system, because TSP-1 is the naturalmediator of apoptosis whose interaction with the IAP and α_(v)β₃ cellreceptors is to be suppressed. It is possible by adding TSP-1 to reachapoptosis rates of 5%, preferably of 7%, particularly preferably of 10%,depending on the concentration of the mediator. However, it is clearthat other substances which interact with α_(v)β₃ and IAP in ananalogous manner to TSP-1 are also suitable according to the invention.An alternative to TSP-1 in the sense referred to above is to increasethe apoptosis rate by simultaneous addition of the RGD and CBD peptides.

These mediators are hormones and other substances with apoptoticactivity. These mediators are added in dissolved form to the cellculture, and it should be noted that the solutions used must be sterile.The sterility of the solutions can be achieved in various ways,preferably by heat treatment (autoclaving at 2 bar and 120° C.) or, ifthis method is unsuitable because of a particular sensitivity of themediator to heat, by sterilizing filtration, for example using Nalgenedisposable sterilizing filters. Methods of this type for sterilizingadditions to cell culture are well known to the skilled worker.

To identify substances having antiapoptotic activity, test substanceswhose effect on the apoptosis of the cultivated cells is to beinvestigated are added to the cell culture. These are preferablymonoclonal antibodies, antibody fragments, polyclonal antibodies andpeptides. However, it is also possible to add other substances suspectedof being able to display an antiapoptotic effect. These substances arepreferably administered in dissolved form. The solvents must in thiscase be compatible with the cell culture. These test substances aretherefore preferably dissolved in buffer solutions, which have generallybecome widely used in cell culture. Examples thereof are phosphatebuffers, sodium carbonate buffers and others. The dissolved testsubstances are preferably sterilized by filtration (sterilized) bysterilizing filtration (Nalgene disposable sterilizing filters) beforeaddition to the cell culture in order to remove contaminatingmicroorganisms or spores of fungi and undissolved constituents.

The dissolved test substance is preferably equilibrated i.e. adjusted tothe temperature of the cell culture, before addition to the cellculture. The volumes depend on the concentration which is to be reachedof the test substance employed, and the volumes are preferably small sothat no dilution effects occur in the cell culture media. The methodswhich can be used to introduce such test substances into cell cultures,preferably in the dissolved state, are well known to the skilled worker.

The decrease in the apoptosis rate can be determined by any suitablemeasurement method. A particularly suitable early indicator ismeasurement of the nonappearance of the calcium influx into the cellthrough use of intracellular calcium indicators. A method suitable fordetermining the exact apoptosis rates is staining of the DNA ofapoptotic cells and subsequent morphometric cell nucleus analysis oranalysis of the cellular DNA content in a flow cytometer. An example ofa suitable fluorescent dye is DAPI. These methods are well known in theprior art and are generally used for detecting apoptotic cells. If thenumber of apoptotic cells decreases after the use of a potentialinhibitor of apoptosis by more than 50%, preferably more than 70% andparticularly preferably by more than 90%, based on the number ofapoptotic cells in the test system in static culture, where appropriateafter addition of an apoptosis-inducing substance, then this substanceis regarded according to the invention as an inhibitor of apoptosis inthe cells used. The nonappearance of the calcium influx into the cellcan be used as early indicator for screening purposes. Substances whichlead to a nonappearance of the calcium influx must subsequently bedetected by DAPI staining.

It is also possible to use other DNA dyes (e.g. Hoechst 33258) orconventional methods such as the TUNEL assay (Tdt-mediated X-dUTP nickend labeling; DNA breaks), the detection of apoptotic enzymes (e.g.PARP, caspases) or proteins (e.g. p53, CD95), the translocation ofphosphatidylserine with fluorescent annexin or the detection of the DNAladder in an agarose gel.

It is possible with the method of the invention to find substances withantiapoptotic activity. These are preferably compounds which bind eitherto receptors on the cell surface, particularly preferably IAP and/or theintegrin α_(v)β₃, or to humoral factors in the blood circulation,particularly preferably TSP-1, in such a way that the specificsimultaneous interaction described herein between TSP-1 and IAP andα_(v)β₃, which leads to induction of apoptosis, does not take place, sothat apoptosis cannot be induced and thus does not occur.

In a further preferred embodiment, these substances bind to IAP and thusprevent the apoptosis-specific calcium influx into the cell, so thatapoptosis cannot be induced and thus does not occur.

These inhibitors are suitable for producing pharmaceutical preparationswhich can be used to treat conditions in which a suppression ofapoptosis is desired, preferably in the vascular system for suppressingapoptosis of endothelial cells and other cells (e.g. smooth musclecells). It is possible in principle to use the substances which haveantiapoptotic activity and can be identified by the method of theinvention to inhibit apoptotic states in all cells which express IAP andα_(v)β₃ on their surface. This makes it possible to treat conditions inwhich an inhibition of apoptosis is indicated, such as, in particular,arteriosclerosis, which leads via inhibition of programmed cell death inthe lesions to an improvement in the state of the vessels. Aprophylactic treatment is also made possible by use of such inhibitorsin pharmaceutical preparations.

Active substances to be tested with the method of the invention are, inparticular, monoclonal antibodies and peptides which can easily beobtained with conventional methods of molecular biology and geneticmanipulation. However, it is clear that other substances which have acorresponding effect can also be found using the test system of theinvention. The term “antibody” is used here to describe both completeantibodies (i.e. antibodies which have two heavy and two light chains)and fragments of antibodies which have an antigen-binding site. Theidentification of an anti-TSP-1 antibody with antiapoptotic activity isdescribed in example 10. It is possible in a similar way to test manyother antibodies or antibody fragments in order to prevent induction ofapoptosis.

It is clear that it is possible to produce a whole series of peptidesand antibodies (antibody fragments) which can be identified as havingantiapoptotic activity using the method of the invention in a similarway. In addition, it is also possible to identify other substances whichare not antibodies (antibody fragments) or peptides as havingantiapoptotic activity with the aid of the method of the invention.

Particularly preferred substances which can be investigated as testsubstances in the test system of the invention are low molecular weightcompounds. Such compounds often have no or only a few side effects ifthey are employed as active principle in a pharmaceutical composition. Afurther advantage of such substances is the possibility of oraladministration.

Examples thereof are cyclic pentapeptides as described by Haubner et.al., J. Am. Chem. Soc. 1996, 118, 7641-7472. The low molecular weightsubstances include according to the invention small peptides, aminoacids and amino acid analogs, steroids, nucleotides and other organicchemical substances with a molecular weight of ≦5 000, preferably ≦3 000and particularly preferably ≦2 000. [Haubner, R., Gratias, R.,Diefenbach, B., Goodman, S. L., Jonczyk, A., Kessler, H., Structural andFunctional Aspects of RGD-Containing Cyclic Pentapeptides as HighlyPotent and Selective Integrin α _(v)β₃ Antagonists, 118, 7461-7472(1996)].

Corresponding products are produced by conventional methods. Forexample, it is possible to dissolve peptides or antibodies (antibodyfragments), which are active ingredients of a pharmaceuticalpreparation, in a pharmaceutically acceptable carrier. One example of apharmaceutically acceptable carrier may be buffer solutions such asphosphate buffer or citrate buffer. It is also possible to maintain theactivity of the peptides by adding reagents which are pharmaceuticallyacceptable and, for example, maintain a reducing environment in thepharmaceutical preparation.

The specific dosage and posology for each patient depends on a number offactors, including the activity of the specific compounds used, the ageof the patient, the body weight, the general state of health, the sex,the diet, the time of administration, the route of administration, therate of excretion, the combination with other medicaments and theseverity of the individual disorder for which the therapy is applied. Itwill be established by his physician as a function of these factors.

Polypeptide medicaments, e.g. protein medicaments or antibodymedicaments, are normally administered parenterally, e.g. by aninhalation spray, rectally, by subcutaneous, intravenous, intramuscular,intraarticular and intrathecal injection or infusion techniques, orexternally in pharmaceutical formulations which comprise conventionalpharmaceutically acceptable carriers, adjuvants and vehicles. Otherroutes of administration are also suitable depending on the nature ofthe identified substance, e.g. orally.

The invention likewise provides pharmaceutical compositions whichcomprise an effective amount of a substance having antiapoptoticactivity in combination with a conventional pharmaceutical carrier. Apharmaceutical carrier is, for example, a solid or liquid filler, anencapsulating material or a solvent. Examples of materials which can beused as pharmaceutical carriers are sugars such as lactose, glucose andsucrose; starch such as corn starch and potato starch; cellulose andderivatives thereof, such as sodium carboxymethylcellulose,ethylcellulose and cellulose cetate; powdered tragacanth; malt; gelatin;tallow; medicament carriers such as cocoa hatter and suppository waxes;oils such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; polyols such as propylene glycol,glycerol, sorbitol, mannitol and polyethylene glycol; esters such asethyl oleate and ethyl laureate; agar; buffering agents such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution, ethyl alcohol and phosphatebuffer solutions as well as other non-toxic compatible substances whichare used in pharmaceutical formulations. Wetting agents, emulsifiers andglidants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, glidants, coating agents and perfuming agents andpreservatives may likewise be present in the preparations according tothe requirements of the pharmaceutical technologist. The amount ofactive ingredient combined with the carrier materials in order toproduce a single dose will vary depending on the treated patient and thespecific method of administration. One example of such a pharmaceuticalformulation is shown in example 13.

ABBREVIATIONS

TAP: Integrin-associated protein (CD 47)

CBD: C-terminal cell-binding domain

TSP-1: Thrombospondin 1

RGD: Arg-Gly-Asp

HUVEC: Human umbilical vein endothelial cells

HPEC: Human placental endothelial cells

DAPI: 4′,6-Diamidino-2-phenylindole

The following figures explain the invention in more detail:

FIG. 1 shows the detection of thrombospondin in the supernatant ofendothelial cells (HUVEC) cultivated under serum-free conditions.Western blot analysis with anti-thrombospondin antibodies. Row 1:serum-free medium; row 2: serum-free medium, statically conditioned for4 days; row 3: 500 ng of TSP-1; row 4: serum-free medium, dynamicallyconditioned for 3 days.

FIG. 2 shows the quantitative detection of the secreted TSP-1 in thesupernatant of statically and dynamically cultivated endothelial cells(HUVEC).

FIG. 3 shows the change in the intracellular calcium level ofendothelial cells in static culture after the use of thrombospondin andthe two active peptides.

FIG. 4 shows the geometry of the flow chamber and of the flow divider inthe perfusion chamber. Endothelial cells grow in the free area.

FIGS. 5 a and 5 b show evaluation grids for experiments with the flowdivider and a position-resolved representation of the occurrence ofapoptosis in the perfusion culture with variable flow conditions. Afterperfusion culture for 2 days, the apoptosis rate is determined for eacharea. Significant apoptosis rates can be detected behind the flowdivider. The apoptosis rate depends on the position in the perfusionchamber.

The following examples explain the invention in more detail.

EXAMPLE 1 Cultivation of Human Endothelial Cells from Umbilical Veins(HUVEC)

Solutions (Sterile):

-   -   Culture medium: IF basal medium+15% (v/v) NCS, 5 μg/m|        transferrin, 5 μg/ml heparin, 0.7 μg/ml FGF, 2 mM L-glutamine        [IF basal medium: 1:1 mixture of Iscove's modified Dulbecco        medium (IMDM) and Ham's F12, both from Life Technologies,        Paisley (England)]    -   NCS: Newborn calf scrum (Sebak, Aidenbach)    -   FGF: Fibroblast growth factor (own preparation purified from pig        brain)

Materials:

-   -   Cell culture vessels, gelatinized

HUVEC are cultivated in gelatin-coated culture vessels at 37° C., 5% CO₂with a water vapor-saturated air atmosphere. The culture medium ischanged every 2-3 days; at confluence, the cells are passaged with adivision rate of from 1:3 to 1:5. HUVEC grow with strict contactinhibition and form monolayer cell lawns with a typical cobblestonemorphology. At confluence, the cultures reach cell densities of 4−9×10⁴cells/cm². Only HUVEC cultures of passages 1-4 are used for apoptosisinvestigations.

Supplement

Coating of Culture Vessels

Solutions (Sterile)

-   -   Gelatin solution, 1% (w/v) in Milli-Q water    -   Suspend 1 g of gelatin (cell culture-tested) in 100 ml of        Milli-Q water, dissolve by autoclaving at 121° C. and 2 bar for        20 min and store at room temperature.    -   PBS (140 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄)        -   8 g/l NaCl        -   0.2 g/l KCl        -   1.44 g/l Na₂HPO₄X₂ H₂O        -   0.2 g/l KH₂PO₄    -   Dissolve the salts in an appropriate volume of Milli-Q water,        autoclave at 121° C. and 2 bar for 20 min and store at room        temperature. The pH is checked and is between 7.2 and 7.4.

Materials:

-   -   Cell Culture Vessels

Procedure:

Culture vessels are coated with gelatin for the cultivation ofadherently growing cells. The base of the cell culture vessels iscovered with sterile gelatin solution, and the cell cultures are left atroom temperature for 15 min. The gelatin solution is aspirated off, thecell culture vessels are washed once with PBS and can be used thus.

Subcultivation of Adherent Cells

Solutions (Sterile)

-   -   PBS    -   Trypsin/EDTA (0.05% (w/v)/0.02% (w/v))        -   0.1 ml of trypsin stock solution        -   0.05 nil of EDTA stock solution    -   Make up to 50 ml with sterile PDS and store in 10 ml portions at        −20° C.

Materials:

Cell culture vessels, gelatinized

Procedure:

All the cell types listed are detached from the culture surface withtrypsin/EDTA solution. The culture medium is aspirated off, the base ofthe culture vessel is briefly washed with PBS and covered withtrypsin/EDTA solution (−1 ml for a 25 cm² culture bottle). The enzymesolution is aspirated off again immediately so that a thin film ofliquid remains on the cells. The cells are left at room temperature for1-10 min, and detachment of the cells is followed under the microscope.The detachment of the cells can be speeded up by gentle tapping of theedge of the culture vessel. The cells are taken up in fresh culturemedium, possibly counted and seeded into new culture vessels.

EXAMPLE 2 Cultivation of Bovine Aorta Endothelial Cells (BAEC)

Solutions (Sterile):

-   -   Culture medium: IF basal medium+5% (v/v) NCS, 5 μg/ml        transferrin, 20 μg/ml heparin, 2 mM L-glutamine; temperature        adjusted to 37° C.

Materials:

-   -   Cell culture vessels, gelatinized

Procedure:

BAEC are cultivated in gelatin-coated culture vessels at 37° C., 5% CO₂with a water vapor-saturated air atmosphere. The culture medium ischanged every 2-3 days; at confluence, the cells are passaged with adivision rate of from 1:3 to 1:5. BAEC grow with strict contactinhibition and form monolayer cell lawns with a typical cobblestonemorphology. The cell density is 7−9×10⁴ cells/cm².

EXAMPLE 3 Cultivation in the Perfusion System

Solutions (Sterile):

-   -   Medium (specifically for the particular cells/HUVEC, see example        1)    -   Milli-Q water (purified water for cell cultures)

Materials:

-   -   Small Petri dish, gelatinized    -   Polycarbonate plug for Petri dishes    -   Silicone sheets    -   Peek screws    -   Silicone tubing ID 0.5 mm    -   Maprene pump tubing ID 1 mm    -   Aluminum plate with fastenings for plug    -   Greiner tubes    -   Schott bottles with Teflon lid seal

Procedure:

The cells are subcultivated as described. After the adherent cells havereached confluence, they can be introduced into the perfusion system. Asystem is assembled for this purpose. All the parts have previously beencleaned thoroughly with Milli-Q water. The completely assembled systemis autoclaved for 40 minutes. The storage vessel consists of a Schottbottle with a special Teflon lid. The lid has 3 holes through whichstainless steel tubes (3 cm, 1 mm ID) are passed. One tube is providedwith a sterilizing filter and serves for pressure equalization. Theother two form the inlet and outlet for the medium. One tube is extendedtoward the inside with silicone tubing so that it dips into the mediumin the bottle. From here, it continues through silicone and Maprene pumptubing to a 15 ml Greiner tube which is likewise closed with a specialTeflon lid through which in turn two stainless steel tubes pass. TheGreiner tube serves for preheating the medium and as an air bubble trap.From here, the medium is passed through further silicone tubing to theplug and to the Peek connector on the upper side of the plug. For thecultivation, the plug is placed on a Petri dish with the relevant cells.The plug is sealed by an O ring on the side. A specially cut siliconesheet is placed on the base of the Petri dish to prevent crushing of thecells. The exit from the plug is in turn coupled by means of a Peekadapter to silicone tubing and returns the medium to the mediumreservoir. Before starting the system, the medium storage vessel and theGreiner tube are charged with preheated medium. The medium in the Petridish is aspirated off from the cells, and the silicone sheets arecautiously placed inside. The plug is then rapidly pressed on, and thesystem can be attached to the peristaltic pump. The pumping rate can beadapted according to the requirements of the cells. It depends on theinternal diameter of the pump tubing and the chosen pump revolutioncount. After a perfusion time which can be chosen unrestrictedly, thesystem can be disassembled in a sterile cabinet, and the cells can beemployed for further tests. The modular design makes diverse uses of thesystem possible. It is possible with the aid of valves and switches fromHPLC technology for substances to be introduced subsequently and forsamples of medium to be removed. It is also possible by using adaptedmedium (e.g. IF medium with 25 HEPES buffer, 12.5 mM NaCl and 0.5 mMsodium carbonate) to work outside an incubator. A hotplate or a heatedchamber is then used for temperature control.

For the experiments with the flow divider, a further silicone sheet isalso placed inside (cf. FIG. 4). It is possible in this way to changethe flow characteristics in the chamber.

EXAMPLE 4 Cultivation in the Cone and Plate Shear Apparatus

Solutions:

-   -   Culture medium with 200 U/ml penicillin, 200 μg/ml streptomycin    -   70% (v/v) ethanol

Material:

-   -   Cone and plate shear apparatus, Bussolori et al. (1982) Rev.        Sci. Instrum. 53, 1851-1854.    -   Gelatinized culture dish (Ø=94 mm

Procedure:

The cone and plate shear apparatus is first cleaned with a soft clothand 70% ethanol, the cone is sterilized, and the apparatus is thenstabilized at 37° C. in a heating cabinet for at least 30 min. Theprecultivated cells are washed with culture medium and provided with0.06 ml/cm² fresh culture medium, and the culture dish is rapidly fittedinto the cone and plate shear apparatus.

The cone is raised using the coarse adjustment, and the culture dishtogether with lid is carefully inserted into the holder providedtherefor. The lid is taken off and the cone is lowered onto the culturedish and adjusted: the tip of a cone is then a minimal distance from thecell lawn and the cone moves round without abrading. The assembled coneand plate shear apparatus is placed in an incubator at 37° C., 5% (v/v)CO₂ with a water vapor-saturated atmosphere for dynamic cultivation ofthe cells.

EXAMPLE 5 Preparation of Conditioned Medium

Solutions (Sterile):

-   -   HUVEC medium

Materials:

-   -   HUVEC, confluent    -   Greiner tubes

Procedure:

HUVEC culture medium from example i is brought onto a confluent celllawn of human umbilical vein endothelial cells and conditioned for 48hours-72 hours. The conditioned medium is then centrifuged at 1000×g for5 minutes. The conditioned medium is frozen at −20° C. until used. Theconditioned HUVEC medium is employed for apoptosis investigations. Forthis purpose it can be supplemented with 2 mM glutamine and 0.7 μg/mlFGF.

EXAMPLE 6 Determination of the Apoptosis Rate by Staining of ApoptoticCells with DAPI

DAPI belongs to the group of indole dyes and is a selective DNA dye. Thedye is excited at 340-360 nm, and the emission maximum is at 480 nm. Itis employed for apoptosis investigations [cf. Cohen et al., ImmunologyToday, 14, No. 3, 126-130 (1993)].

Morphological Evaluation:

Solutions:

-   -   PBS    -   Formaldehyde solution 4% (v/v) formaldehyde in PBS    -   DAPI solution (Molecular Probes, Leiden, The Netherlands) 2        μg/ml DAPI in metanol

Materials:

-   -   Petri dish (35 mm) with cells in culture

Procedure:

The culture supernatant is aspirated off of a Petri dish, and the celllawn is fixed with 1 ml of formaldehyde solution on ice for 15 minutes,washed twice with 2 ml of PBS, treated with 0.5 ml of DAPI solution for15 minutes, washed with PBS and evaluated under a fluorescencemicroscope. The UV filter set and a 20× or 40× objective are used. 500-1000 cells are selected at random, and the cells with apoptotic nucleiare counted.

The apoptosis index is calculated by the following formula:

${{Apoptosis}\mspace{14mu} {{index}\mspace{14mu}\lbrack\%\rbrack}} = {\frac{{number}\mspace{14mu} {of}\mspace{14mu} {apoptotic}\mspace{14mu} {cells}}{{total}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {cells}} \cdot 100}$

Flow Cytometry:

Solutions:

-   -   PBS    -   Medium    -   Ethanol (analytical reagent ice-cooled (−20° C.)    -   DAPI buffer    -   DAPI stock solution    -   DAPI stain solution

Procedure:

The culture supernatant is aspirated off, and the cells are trypsinizedwithout being washed with PBS. The cell suspension is taken up inmedium, counted and centrifuged at 800×g for 5 minutes, and the sedimentis resuspended in 0.5 ml of IF and added dropwise to 1.5 ml of ice-coldethanol. The suspension is stored at −20° C. overnight. Renewedcentrifugation and resuspension of the sediment in 2 ml of PBS arefollowed by incubation at 37° C. for half an hour, a furthercentrifugation, resuspension of the sediment in 5 ml of DAPI solutionand counting in a flow cytometer at a counting rate of 50-300 events persecond. The resulting plot shows a large peak of cells in the G₁ phaseof the cell cycle, followed by a fraction of cells in the S phase(medium fluorescence intensities) and a last peak of high fluorescenceintensities, which represents the cells in the G₂ phase. Apoptotic cellsappear, owing to the decreasing absolute amount of DNA per cell, in asub-G₁ peak [Darzynklewicz Z. et al., Cytometry, 13, 795-808 (1992);Zamai L. et al., Cytometry, 14, 891-897 (1993)]. This shows theoccurrence of apoptosis under the chosen conditions.

EXAMPLE 7 Measurement of the Induced Calcium Influx into the CellsThrough Use of the Intracellular Calcium Indicator Fluo-3 AM

Fluo-3 is a calcium indicator which forms a fluorescent complex afterbinding of Ca²⁺. The ester Fluo-3 AM is taken up by the cells throughdiffusion. The calcium indicator Fluo 3 is produced only afterhydrolysis of the ester in the cell. Extracellular dye ester thus doesnot impair the measurement. The measurement is carried out with normalfluorescein filters. At an excitation wavelength of 488 nm (500 nm), theemission maximum is at 525 nm and increases by a factor of 100 (200)through calcium binding. The measurement range is between 0.05 and 20 μMfree Ca²⁺ (Merritt, L E. et al., Biochem. J. 269, 513-519 (1990).

Solutions:

-   -   PBS    -   Medium    -   Fluo-3 AM stock solution    -   50 μg in 10 μl of DMSO dimethyl sulfoxide (Molecular Probes,        Leiden) (6 mM)    -   Fluo-3 AM concentration used    -   3 μM in serum-free medium

Materials:

-   -   Cells in culture

Procedure:

Confluent HUVEC cell lawns in 24-well plates are washed three times withserum-free medium, incubated with preheated serum-free medium withFluo-3 AM under culture conditions for 30 minutes and washed threetimes, and the plate is measured with a fluorimeter. First thesensitivity of the instrument is adjusted, and then fluorescence valuescorresponding to the instantaneous calcium level in the cell arerecorded every 1-10 seconds. Substances can be added to the cells whilethe measurement is taking place. If a substance has an effect on thecalcium level, this can be recognized from the increase or decrease inthe fluorescence values (see FIG. 3). Assessment of the calcium level ispossible with this method. The calcium influx is an early sign ofapoptosis taking place.

EXAMPLE 8 Induction of Apoptosis in Cultivated Endothelial Cells withTSP-1

The cells are cultivated as described in examples 1 and 2. Aftercomplete confluence is reached, the cells are employed for the test.Firstly, thrombospondin 1 is added in various concentrations (1-600μg/ml) to fresh medium and compared with the effect of conditionedmedium on the spontaneous apoptosis rate of HUVEC. The following tableshows that addition of thrombospondin leads to a significant increase inthe apoptosis rate. It corresponds to that achievable with conditionedmedium. The apoptosis observed on addition of fresh medium is induced bythe thrombospondin secreted during the experiment. Thrombospondin has noeffect on perfused HUVEC. The apoptosis rate is determined by stainingapoptotic cells with DAPI.

TABLE 3 Culturing Apoptosis rate (%) conditions Culture medium after 24h Static Fresh medium 0.9 ± 0.1 Static Conditioned 3.0 ± 0.2 mediumStatic Fresh + TSP-1 3.0 ± 0.4 (1 μg/ml) Dynamic Fresh medium <0.1Dynamic Conditioned <0.1 medium Dynamic Fresh TSP-1 <0.1 (1 μg/ml)

The apoptosis rate has been indicated after DAPI staining as shown inexample 6 (the mean and the standard deviation in each case areindicated). The percentage apoptosis rate is based on the total cellcount.

EXAMPLE 9 Test System for Peptides or Proteins with AntiapoptoticActivity

The cells are cultivated as described in examples 1 and 2. The cells areseeded into the appropriate culture vessels (e.g. 24-well plate/0.5 mlper well) and employed for the test after complete confluence isreached. Investigations into the effect of various peptides and proteins(table 2) on the apoptosis rate of endothelial cells are carried out.For this purpose, the peptides and proteins (table 4) are dissolved inculture medium (example 1) and employed in the stated concentrations(table 4). The medium with the dissolved samples is added to the cellsand incubated under culture conditions (example 1) for three days. Thecells are then stained with DAPI to determine the apoptosis rate asdescribed in example 6. The results of 3 independent experiments arecompiled in table 2, stating the means and the standard deviation. Theapoptosis-inducing effect of conditioned medium, TSP-1 and of active RGDpeptide together with the CBD peptide is clearly evident.

Intracellular calcium measurement (example 7) can be employed as earlyindicator. For this purpose, the cells are first pretreated as describedin example 7 and measured in a fluorimeter at the time of addition ofthe peptides or of TSP-1. Only TSP-1 or simultaneous addition of bothpeptides leads to an increase in the intracellular calcium level (FIG.3). The addition of fresh medium or of only one peptide has no effect onthe calcium level. The influx of calcium into the cells can be employedas early indicator. Verification of positive substances (calcium signal)can be obtained by determining the apoptosis rate with DAPI (example 6)after incubation for at least 24 hours. It is thus possible to identifycompounds with antiapoptotic activity rapidly and unambiguously.

TABLE 4 Incubation Biological Ligand Concentration time activityThrombospondin 1 μg/ml (2-)24-72 h binds integrin α_(v)β₃ and IAP RGD250 μg/ml (2-)24-72 h binds cyclic, act. integrin α_(v)β₃ RGD 250 μg/ml(2-)24-72 h Inactive cyclic, inact. IAP peptide 400 μg/ml (2-)24-72 hbinds IAP (CBD)

EXAMPLE 10 Identification of an Antibody with Antiapoptotic ActivityUsing the Method of the Invention

The cells are cultivated as described in examples 1 and 2. The cells areseeded in the appropriate culture vessels (e.g. 24-well plate/0.5 ml perwell) and employed for the test after complete confluence is reached.The cells are supplied with new medium: (a) fresh culture medium [basicapoptosis rate], (b) conditioned medium (example 5) [apoptosis-inducingeffect], (c) conditioned medium with rabbit anti-TSP-1 antiserum (1:20)[apoptosis-inducing and apoptosis-inhibiting substances], (d) freshculture medium with rabbit anti-TSP-1 antiserum (1:20)[apoptosis-inhibiting substance], (e) fresh medium with 1 μg/ml TSP-1[apoptosis-inducing substance], (f) fresh medium with 1 μg/ml TSP-1 andrabbit anti-TSP-1 antiserum (1:20) [apoptosis-inducing andapoptosis-inhibiting substances], (g) fresh medium with monoclonalanti-TSP-1 antibody (1:30) [apoptosis-inhibiting substance]. Afterincubation under culture conditions (example 1) for 24 h, the cells arefixed, stained with DAPI and examined morphologically under afluorescence microscope or investigated by flow cytometry (example 6).The apoptotic cells and the total cell count are determined and theapoptosis index is calculated (percent of apoptotic cells). The data of3 independent experiments are indicated in table 1, stating the mean andstandard deviation.

The experiments with polyclonal antiserum (=antiserum: polyclonalantibody against TSP-1 (from rabbits) unpurified; protein concentration:900 μg/ml) and with a monoclonal antibody against TSP-1 (=mAb:monoclonal antibody against TSP-1M 101 unpurified; proteinconcentration: 300 μg/ml) show a significant decrease in the apoptosisrate (>50%) and can be employed as inhibitors of naturalapoptosis-inducing substances (conditioned medium and TSP-1). It wasthus possible to identify them as antiapoptotic. The antiserum and themonoclonal antibody were provided by Prof. P. Vischer, Institut fürArterioskleroseforschung Münster.

The test system can also be carried out with the calcium indicatorFluo-3 AM. For this purpose, the cells are likewise cultivated asdescribed in examples 1 and 2. The cells are employed for the test aftercomplete confluence is reached. They are loaded with a calcium indicator(example 7) placed in a fluorimeter and measured. During themeasurement, the substances b, c, e, f, g are added to the cells. Anactivator (conditioned medium or TSP-1) is present in each of theseexamples. In examples b) and e) there is an influx of calcium and thusan increase in the measured fluorescence values. If apoptosis-inducingsubstances are added at the same time, as in example c), f) and g), noincrease can be observed. The monoclonal antibody against TSP-1 or thepolyclonal antiserum prevent the influx of calcium and the induction ofapoptosis. It is thus possible by simultaneous addition of an activator(e.g. TSP-1) and of the substance to be tested to establish a rapid testmethod for potential apoptosis inhibitors.

Two further antibodies which can be employed as apoptosis inhibitorswere identified with this method with the described method. These are:

-   -   1. Monoclonal antibody against integrin α_(v) supplied by        Chemicon (No. MAB 1960), clone VNR139 (100 μl); dilution        employed: 1:100 (mAb/α_(v))    -   2. Monoclonal antibody against human IAP (CD 47) supplied by        CYMBUS Biotechnology (No. CBL 489), clone BRIC 126,        concentration: 100 μg/ml; dilution employed: 1:100 (1 μg/ml)        (mAb/TAP)

The antibodies were added to the cells after confluence was reached withthe activator TSP-1 (1 μg/ml) in the stated concentrations. Afterincubation under static conditions for 48 hours, the following apoptosisrates were measured:

TABLE 1 Culture medium Apoptosis rate [%] (with SEM) Fresh medium +TSP-1 7.3 ± 0.6 Fresh medium + TSP-1 + mAb/a_(v) 0.9 ± 0.2 Freshmedium + TSP-1 + mAb/IAP 0.5 ± 0.1 Apoptosis Culture rate conditionsCulture medium (24 h) (%) Static Fresh medium (a) 0.9 ± 0.1 StaticConditioned medium (b) 3.0 ± 0.2 Static Conditioned + anti-TSP 1 (c)0.1 ± 0.1 Static Fresh + TSP 1 (e) 3.0 ± 0.4 Static Fresh + anti-TSP 1(d) 0.2 ± 0.1 Static Fresh + TSP I + anti-TSP 1 (f) 0.2 ± 0.1 StaticFresh + mAb TSP 1 (g) 0.4 ± 0.1

EXAMPLE 11 Suppression of the Apoptosis Induced by Static CultureConditions Through Complexation of the Calcium or Inhibition of theCalcium Influx

Material (in Addition to Example 1):

Culture medium with 2.5 mmol/l EGTA

Procedure:

The cells are prepared as described in example 1 and, after confluenceis reached, cultivated in the cone and plate shear apparatus (seeexample 4) for a further 24 hours. The culture dish is removed anddivided into 12 independent culture wells (culture area of each well 0.8cm²) by putting in a suitable insert. Half of the culture is cultivatedin culture medium with EGTA and the second half is cultivated in culturemedium without EGTA. Table 5 below shows that the result of cultivationin culture medium with EGTA is a significant reduction in the apoptosisrate, i.e. complexation of the calcium with EGTA leads to an inhibitionof apoptosis. The apoptosis rate is determined by staining the apoptoticcells with DAPI.

TABLE 5 Duration of Apoptosis rate Apoptosis rate experiment [hours] (%)with EGTA [2.5 mM] (%) without EGTA 0 0.40 ± 0.22 0.42 ± 0.23 2 0.42 ±0.22 2.22 ± 0.55 3 0.96 ± 0.27 2.80 ± 0.64 4 1.56 ± 0.23 3.10 ± 0.31 52.26 ± 0.35 5.33 ± 0.59

The apoptosis rate was found by the method of DAPI staining as shown inexample 6 of the main patent (the mean and the standard deviation areindicated in each case). The percentage apoptosis rate is based on thetotal cell count.

EXAMPLE 12 Suppression of the Apoptosis Induced by the BiologicallyActive Peptides Through Complexation of the Calcium

Material (in Addition to Example 4):

-   -   Culture medium with biologically active peptides (compare main        patent example 10, table 4)

Procedure:

The cells are prepared as described in example 1 and, after completeconfluence is reached, cultivated in the cone and plate shear apparatus(see example 4) for a further 24 hours. The culture dish is removed fromthe shear apparatus and divided into 12 independent culture wells byputting in a suitable insert as already described in example 11. Thebiologically active peptides are dissolved on the one hand in culturemedium with 0.5 mM EGTA and on the other hand in culture medium withoutEGTA (concentrations: RGD peptide: 250 μg/ml [0.5 mM]; IAP peptide: 400μg/ml [0.5 mM]). Thereafter half of the samples is treated with culturemedium with EGTA and the active peptides and the other half is treatedwith culture medium which contains the active peptides but no EGTA. Theresult of these investigations is shown in table 6 below:

TABLE 6 Duration of Apoptosis rate (%) Apoptosis rate experiments withEGTA and with (%) without EGTA and [hours] peptides with peptides 0 0.45± 0.24 0.61 ± 0.22 2 1.00 ± 0.29 2.08 ± 0.22 3 0.65 ± 0.24 2.67 ± 0.18 41.25 ± 0.33 3.82 ± 0.50

Addition of the biologically active peptides in a culture medium withoutEGTA leads to a marked increase in apoptosis over the period of theexperiment. Addition of EGTA in the culture medium with the activepeptides makes it possible to achieve a significant reduction in theapoptosis rate.

The apoptosis rate was found by the method of DAPI staining as shown inexample 6 of the main patent (the mean and the standard deviation areindicated in each case). The percentage apoptosis rate is based on thetotal cell count.

EXAMPLE 13 Use of Antibodies or Peptides as Active Substance inPharmaceutical Formulation

The compounds with antiapoptotic activity identified in example 10, i.e.the anti-TSP-1 antibody, the monoclonal antibody VNR 139 and themonoclonal antibody BRIC 126, might be employed as active substances inpharmaceutical formulations.

These antibodies are for this purpose expediently employed in aconcentration of 3-5 mg per ml in the following formulation:

-   -   Water for injections    -   Polysorbate 80    -   Disodium hydrogen phosphate/sodium dihydrogen phosphate    -   Sodium chloride

This formulation is administered as solution for injection.

1. A method for treating vascular disorders, the method comprisingadministering to a subject in need of such treatment an effective amountof a composition comprising a pharmaceutically acceptable carrier and ananti-CD47 antibody that binds to at least one compound selected from thegroup consisting of TSP-1, CD47 and α_(v)β₃ in such a way thatsimultaneous binding of TSP-1 to CD47 or α_(v)β₃ is inhibited.
 2. Themethod of claim 1, wherein the anti-CD47 antibody inhibits thesimultaneous binding of TSP-1 to CD47 or integrin α_(v)β₃ on surfaces ofendothelial cells or corresponding recombinant cells.
 3. The method ofclaim 2, wherein the endothelial cells are selected from the groupconsisting of human placental endothelial cells (HPEC), bovine aortaendothelial cells (BAEC), porcine brain capillary endothelial cells(PBMEC), hybrid cell lines, and human microvascular endothelial cells.4. The method of claim 1, wherein the anti-CD47 antibody inhibits thesimultaneous binding of TSP-1 to CD47 or integrin α_(v)β₃ on surfaces ofsmooth muscle cells or fibroblasts.
 5. The method of claim 1, whereinthe anti-CD47 antibody is BRIC
 126. 6. A method for treating vasculardisorders, the method comprising administering to a subject in need ofsuch treatment an effective amount of a composition comprising apharmaceutically acceptable carrier and an anti-CD47 antibody which canbe identified by a method comprising (a) culturing cells which expressboth CD47 and the integrin (b) (i) causing the cells to produce anapoptosis-inducing substance, (ii) adding a substance whichsimultaneously binds CD47 and the integrin α_(v)β₃, by culturing understatic or turbulent flow conditions, thereby inducing apoptosis, or(iii) both (i) and (ii), (c) adding the anti-CD47 antibody, and (d)determining a change in apoptosis rate by determining the portion ofcells with apoptotic nuclei, wherein the anti-CD47 antibody binds to acalcium channel in the cell membrane in such a way thatapoptosis-specific calcium influx into the cell is suppressed.
 7. Themethod of claim 6, wherein CD47 is involved in the production of thecalcium channel.
 8. The method of claim 6, wherein the anti-CD47antibody inhibits apoptosis-specific calcium influx into endothelialcells or corresponding recombinant cells.
 9. The method of claim 8,wherein the endothelial cells are selected from the group consisting ofhuman umbilical vein endothelial cells (HUVEC), human placentalendothelial cells (HPEC), bovine aorta endothelial cells (BAEC), porcinebrain capillary endothelial cells (PBMEC), hybrid cell lines, and humanmicrovascular endothelial cells (HMVEC).
 10. The method of claim 6,wherein the anti-CD47 antibody inhibits apoptosis-specific calciuminflux into smooth muscle cells or fibroblasts.